Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments
TL;DR: This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.
Abstract: Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.
Citations
More filters
TL;DR: This review presents the current research on exopolysaccharides of oral microbes regarding their biosynthesis, regulation, contributions to biofilm formation and stability of the matrix, and immune evasion, as well as a potential nutrient source for species within a biofilm.
Abstract: The oral cavity contains a rich consortium of exopolysaccharide-producing microbes. These extracellular polysaccharides comprise a major component of the oral biofilm. Together with extracellular proteins, DNA, and lipids, they form the biofilm matrix, which contributes to bacterial colonization, biofilm formation and maintenance, and pathogenesis. While a number of oral microbes have been studied in detail with regard to biofilm formation and pathogenesis, the exopolysaccharides have been well characterized for only select organisms, namely Streptococcus mutans and Aggregatibacter actinomycetemcomitans. Studies on the exopolysaccharides of other oral organisms, however, are in their infancy. In this review, we present the current research on exopolysaccharides of oral microbes regarding their biosynthesis, regulation, contributions to biofilm formation and stability of the matrix, and immune evasion. In addition, insight into the role of exopolysaccharides in biofilms is highlighted through the evaluation of emerging techniques such as pH probing of biofilm colonies, solid-state nuclear magnetic resonance for macromolecular interactions within biofilms, and super-resolution microscopy analysis of biofilm development. Finally, exopolysaccharide as a potential nutrient source for species within a biofilm is discussed.
56 citations
Cites background from "Genes Contributing to Porphyromonas..."
...Recently, P. gingivalis strains that were deficient in either the SinR regulator or genes associated with capsule production were less fit in an abscess model of infection or in the presence of mammalian cells, highlighting the importance of capsule in virulence (Miller et al. 2017)....
[...]
TL;DR: The relationship among P.gingivalis, periodontal disease, and digestive tract tumors must be clarified through a multi-center, prospective, large-scale study.
Abstract: Bacterial infection may be involved in the entire process of tissue carcinogenesis by directly or indirectly affecting the occurrence and development of tumors. Porphyromonas gingivalis (P.gingivalis) is an important pathogen causing periodontitis. Periodontitis may promote the occurrence of various tumors. Gastrointestinal tumors are common malignant tumors with high morbidity, high mortality, and low early diagnosis rate. With the rapid development of molecularbiotechnology, the role of P.gingivalis in digestive tract tumors has been increasingly explored. This article reviews the correlation between P.gingivalis and gastrointestinal cancer and the pathogenesis of the latter. The relationship among P.gingivalis, periodontal disease, and digestive tract tumors must be clarifiedthrough a multi-center, prospective, large-scale study.
30 citations
TL;DR: Microarray data obtained from the chronic infection model of immortalized oral epithelial cells that were persistently exposed to P. gingivalis were analyzed to identify hub genes, major biological processes, upstream regulators and genes potentially involved in tumor initiation and progression that might facilitate the prevention and treatment of oral squamous cell carcinoma.
Abstract: Recent investigations revealed the relationship between chronic periodontitis, Porphyromonas gingivalis and cancer. However, host genes that change in response to chronic infection with P. gingivalis and may contribute to oral cancer have remained largely unknown. In the present study, we aimed to comprehensively analyze microarray data obtained from the chronic infection model of immortalized oral epithelial cells that were persistently exposed to P. gingivalis for 15 weeks. Using protein-protein interaction (PPI) networks and Ingenuity Pathway Analysis (IPA), we identified hub genes, major biological processes, upstream regulators and genes potentially involved in tumor initiation and progression. We also validated gene expression and demonstrated genetic alteration of hub genes from clinical samples of head and neck cancer. Overall, we utilized bioinformatical methods to identify IL6, STAT1, LYN, BDNF, C3, CD274, PDCD1LG2, and CXCL10 as potential candidate genes that might facilitate the prevention and treatment of oral squamous cell carcinoma (OSCC), the most common type of head and neck squamous cell carcinoma (HNSCC).
23 citations
TL;DR: Genetic differences between CP3 and H3 are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3.
Abstract: Porphyromonas gingivalis has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two P. gingivalis strains, one from a patient exhibiting severe chronic periodontitis (CP3) and another from a periodontally healthy individual (H3). We previously showed that CP3 and H3 exhibit differences in virulence since H3 showed a lower resistance to cationic peptides compared with CP3, and a lower ability to induce proliferation in gingival epithelial cells. Here, we aimed to determine whether differences in virulence between these two strains are associated with the presence or absence of specific genes encoding virulence factors. We sequenced the whole genomes of both P. gingivalis CP3 and H3 and conducted a comparative analysis regarding P. gingivalis virulence genetic determinants. To do so, we performed a homology search of predicted protein sequences in CP3 and H3 genomes against the most characterized virulence genes for P. gingivalis available in the literature. In addition, we performed a genomic comparison of CP3 and H3 with all the 62 genomes of P. gingivalis found in NCBI's RefSeq database. This approach allowed us to determine the evolutionary relationships of CP3 and H3 with other virulent and avirulent strains; and additionally, to detect variability in presence/absence of virulence genes among P. gingivalis genomes. Our results show genetic variability in the hemagglutinin genes. While CP3 possesses one copy of hagA and two of hagC, H3 has no hagA and only one copy of hagC. Experimentally, this finding is related to lower in vitro hemmaglutination ability of H3 compared to CP3. Moreover, while CP3 encodes a gene for a major fimbrium subunit FimA type 4 (CP3_00160), H3 possess a FimA type 1 (H3_01400). Such genetic differences are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3. Therefore, here we provide new results on the genome sequences, comparative genomics analyses, and phenotypic analyses of two P. gingivalis strains. The genomics comparison of these two strains with the other 62 genomes included in the analysis provided relevant results regarding genetic determinants and their association with P. gingivalis virulence.
17 citations
Cites background from "Genes Contributing to Porphyromonas..."
...HagA is involved in adhesion to both gingival epithelial and endothelial cells (Bélanger et al., 2012); as well as contributing to bacterial survival in both epithelial cells in vitro and murine abscesses (Miller et al., 2017)....
[...]
...Conversely, HagB and HagC do not contribute to P. gingivalis virulence in vivo (Miller et al., 2017), although HagB has been associated with bacterial adhesion in vitro (Song et al., 2005)....
[...]
...gingivalis virulence in vivo (Miller et al., 2017), although HagB has been associated with bacterial adhesion in vitro (Song et al....
[...]
..., 2012); as well as contributing to bacterial survival in both epithelial cells in vitro and murine abscesses (Miller et al., 2017)....
[...]
TL;DR: It is shown that Porphyromonas gingivalis, a Gram-negative periodontal pathogen, expresses a PHP protein, Php1, with divalent metal ion-dependent tyrosine phosphatase activity, a unique configuration in Gram-negatives which may allow P. gedivalis to maintain phosphorylation/dephosphorylation homeostasis in multispecies communities.
Abstract: Protein-tyrosine phosphorylation in bacteria plays a significant role in multiple cellular functions, including those related to community development and virulence. Metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are widespread in Gram-positive bacteria. Here, we show that Porphyromonas gingivalis, a Gram-negative periodontal pathogen, expresses a PHP protein, Php1, with divalent metal ion-dependent tyrosine phosphatase activity. Php1 tyrosine phosphatase activity was attenuated by mutation of conserved histidine residues that are important for the coordination of metal ions and by mutation of a conserved arginine residue, a key residue for catalysis in other bacterial PHPs. The php1 gene is located immediately downstream of the gene encoding the bacterial tyrosine (BY) kinase Ptk1, which was a substrate for Php1 in vitro Php1 rapidly caused the conversion of Ptk1 to a state of low tyrosine phosphorylation in the absence of discernible intermediate phosphoforms. Active Php1 was required for P. gingivalis exopolysaccharide production and for community development with the antecedent oral biofilm constituent Streptococcus gordonii under nutrient-depleted conditions. In contrast, the absence of Php1 had no effect on the ability of P. gingivalis to form monospecies biofilms. In vitro, Php1 enzymatic activity was resistant to the effects of the streptococcal secreted metabolites pABA and H2O2, which inhibited Ltp1, an enzyme in the low-molecular-weight (LMW) phosphotyrosine phosphatase family. Ptk1 reciprocally phosphorylated Php1 on tyrosine residues 159 and 161, which independently impacted phosphatase activity. Loss of Php1 rendered P. gingivalis nonvirulent in an animal model of periodontal disease. Collectively, these results demonstrate that P. gingivalis possesses active PHP and LMW tyrosine phosphatases, a unique configuration in Gram-negatives which may allow P. gingivalis to maintain phosphorylation/dephosphorylation homeostasis in multispecies communities. Moreover, Php1 contributes to the pathogenic potential of the organism.IMPORTANCE Periodontal diseases are among the most common infections of humans and are also associated with systemic inflammatory conditions. Colonization and pathogenicity of P. gingivalis are regulated by signal transduction pathways based on protein tyrosine phosphorylation and dephosphorylation. Here, we identify and characterize a novel component of the tyrosine (de)phosphorylation axis: a polymerase and histindinol phosphatase (PHP) family enzyme. This tyrosine phosphatase, designated Php1, was required for P. gingivalis community development with other oral bacteria, and in the absence of Php1 activity P. gingivalis was unable to cause disease in a mouse model of periodontitis. This work provides significant insights into the protein tyrosine (de)phosphorylation network in P. gingivalis, its adaptation to heterotypic communities, and its contribution to colonization and virulence.
16 citations
Cites background from "Genes Contributing to Porphyromonas..."
...Additionally, in a recent study using a random transposon insertion library, we found that insertional inactivation of either php1 or ptk1 significantly reduced fitness in an epithelial colonization model (54), indicating that the P....
[...]
References
More filters
TL;DR: Policy makers need to be aware of a predictable increasing burden of SP due to the growing world population associated with an increasing life expectancy and a significant decrease in the prevalence of total tooth loss throughout the world from 1990 to 2010.
Abstract: We aimed to consolidate all epidemiologic data about severe periodontitis (SP) and, subsequently, to generate internally consistent prevalence and incidence estimates for all countries, 20 age groups, and both sexes for 1990 and 2010. The systematic search of the literature yielded 6,394 unique citations. After screening titles and abstracts, we excluded 5,881 citations as clearly not relevant to this systematic review, leaving 513 for full-text review. A further 441 publications were excluded following the validity assessment. A total of 72 studies, including 291,170 individuals aged 15 yr or older in 37 countries, were included in the metaregression based on modeling resources of the Global Burden of Disease 2010 Study. SP was the sixth-most prevalent condition in the world. Between 1990 and 2010, the global age-standardized prevalence of SP was static at 11.2% (95% uncertainty interval: 10.4%-11.9% in 1990 and 10.5%-12.0% in 2010). The age-standardized incidence of SP in 2010 was 701 cases per 100,000 person-years (95% uncertainty interval: 599-823), a nonsignificant increase from the 1990 incidence of SP. Prevalence increased gradually with age, showing a steep increase between the third and fourth decades of life that was driven by a peak in incidence at around 38 yr of age. There were considerable variations in prevalence and incidence between regions and countries. Policy makers need to be aware of a predictable increasing burden of SP due to the growing world population associated with an increasing life expectancy and a significant decrease in the prevalence of total tooth loss throughout the world from 1990 to 2010.
1,346 citations
"Genes Contributing to Porphyromonas..." refers background in this paper
...While the organism is usually in mutualistic balance with the host, breakdown of homeostasis and the induction of dysbiotic host responses leads to chronic periodontal diseases, one of the most common infectious diseases of humans worldwide (Kassebaum et al., 2014; Hajishengallis and Lamont, 2016)....
[...]
TL;DR: Although periodontal disease is localized to the tissues surrounding the tooth, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to delivery of preterm infants.
Abstract: Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. An opportunistic pathogen, P. gingivalis can also exist in commensal harmony with the host, with disease episodes ensuing from a shift in the ecological balance within the complex periodontal microenvironment. Colonization of the subgingival region is facilitated by the ability to adhere to available substrates such as adsorbed salivary molecules, matrix proteins, epithelial cells, and bacteria that are already established as a biofilm on tooth and epithelial surfaces. Binding to all of these substrates may be mediated by various regions of P. gingivalis fimbrillin, the structural subunit of the major fimbriae. P. gingivalis is an asaccharolytic organism, with a requirement for hemin (as a source of iron) and peptides for growth. At least three hemagglutinins and five proteinases are produced to satisfy these requirements. The hemagglutinin and proteinase genes contain extensive regions of highly conserved sequences, with posttranslational processing of proteinase gene products contributing to the formation of multimeric surface protein-adhesin complexes. Many of the virulence properties of P. gingivalis appear to be consequent to its adaptations to obtain hemin and peptides. Thus, hemagglutinins participate in adherence interactions with host cells, while proteinases contribute to inactivation of the effector molecules of the immune response and to tissue destruction. In addition to direct assault on the periodontal tissues, P. gingivalis can modulate eucaryotic cell signal transduction pathways, directing its uptake by gingival epithelial cells. Within this privileged site, P. gingivalis can replicate and impinge upon components of the innate host defense. Although a variety of surface molecules stimulate production of cytokines and other participants in the immune response, P. gingivalis may also undertake a stealth role whereby pivotal immune mediators are selectively inactivated. In keeping with its strict metabolic requirements, regulation of gene expression in P. gingivalis can be controlled at the transcriptional level. Finally, although periodontal disease is localized to the tissues surrounding the tooth, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to delivery of preterm infants.
983 citations
"Genes Contributing to Porphyromonas..." refers background in this paper
...HagA can promote attachment to both epithelial and endothelial cells, and antibodies to the hemagglutinin domain are protective in animal models of oral infection (Frazer et al., 2006; Belanger et al., 2012)....
[...]
...RgpA and Kgp also possess haemagglutinin domains homologous to those of HagA (Potempa et al., 1997; Lamont and Jenkinson, 1998; Fitzpatrick et al., 2009; Guo et al., 2010)....
[...]
...HagA is a large protein with a predicted molecular mass of 283.3 kDa and containing multiple contiguous direct repeats of 440–456 amino acids, each of which has hemagglutinin activity (Han et al., 1996)....
[...]
...gingivalis relies on proteolytic activity to produce peptides from proteins as both a carbon and nitrogen source (Lamont and Jenkinson, 1998; Guo et al., 2010)....
[...]
...Our finding that disruption of hagA was deleterious for survival in epithelial cells and murine abscesses is consistent with the documented properties of HagA, and suggest a role for this protein in P. gingivalis pathogenicity....
[...]
TL;DR: In gram-negative bacteria, it is proposed that biosynthesis and translocation of capsular polysaccharides to the cell surface are temporally and spatially coupled by multiprotein complexes that span the cell envelope.
Abstract: Capsules are protective structures on the surfaces of many bacteria. The remarkable structural diversity in capsular polysaccharides is illustrated by almost 80 capsular serotypes in Escherichia coli. Despite this variation, the range of strategies used for capsule biosynthesis and assembly is limited, and E. coli isolates provide critical prototypes for other bacterial species. Related pathways are also used for synthesis and export of other bacterial glycoconjugates and some enzymes/processes have counterparts in eukaryotes. In gram-negative bacteria, it is proposed that biosynthesis and translocation of capsular polysaccharides to the cell surface are temporally and spatially coupled by multiprotein complexes that span the cell envelope. These systems have an impact on both a general understanding of membrane trafficking in bacteria and on bacterial pathogenesis.
946 citations
"Genes Contributing to Porphyromonas..." refers background in this paper
...Production of extracellular polysaccharide can be controlled by a tyrosine phosphatase (Ltp1) and a tyrosine kinase (Ptk1) (Maeda et al., 2008; Wright et al., 2014) which participate in a secretion system homologous to the Wzy-dependent mechanism in E. coli (Whitfield, 2006)....
[...]
TL;DR: This paper presents a meta-modelling study of the regulation of metabolic flow in E. coli through the pathways of Arginine, Citrulline, and Ornithine and concludes that these pathways are dominated by bacteria with extensive repression control.
666 citations
"Genes Contributing to Porphyromonas..." refers background in this paper
...PGN_1444 is annotated as a carbamoyl phosphate synthetase, an enzyme that catalyzes the ATP-dependent synthesis of carbamoyl phosphate from glutamine or ammonia, an important early step in the synthesis of pyrimidine and citrulline as a precursor of arginine (Cunin et al., 1986)....
[...]
TL;DR: This work used massively parallel sequencing to monitor the relative abundance of tens of thousands of transposon mutants of a saccharolytic human gut bacterium, Bacteroides thetaiotaomicron, as they established themselves in wild-type and immunodeficient gnotobiotic mice, in the presence or absence of other human gut commensals.
630 citations
"Genes Contributing to Porphyromonas..." refers methods in this paper
...Briefly, a saturated transposon library was generated using a mariner transposon system (Goodman et al., 2009) in P....
[...]
...Briefly, a saturated transposon library was generated using a mariner transposon system (Goodman et al., 2009) in P. gingivalis 33277....
[...]